Inactivation of CACNA1H induces cell apoptosis by initiating endoplasmic reticulum stress in glioma

Abstract Background Ca2+ channels are abnormally expressed in various tumor cells and are involved in the progression of human glioma. Here, we explored the role of a calcium channel, voltage-dependent, T-type, alpha 1H subunit (CACNA1H), which encodes T-type Ca2+ channel Cav3.2 in glioma cells. Methods Cell viability and apoptosis were detected using cell-counting kit-8 and flow cytometry, respectively. The expression of target protein was determined using western blot analysis. Results Cell viability of U251 cells was inhibited significantly after the knockdown of CACNA1H. The apoptosis of U251 cells was enhanced significantly after the knockdown of CACNA1H. Importantly, knockdown of CACNA1H decreased the levels of p-PERK, GRP78, CHOP, and ATF6, indicating that CACNA1H knockdown activated endoplasmic reticulum stress (ERS) in U251 cells. In addition, T-type Ca2+ channel inhibitor NNC55-0396 also induced apoptosis through the activation of ERS in U251 cells. ERS inhibitor UR906 could block CACNA1H inhibitor ABT-639-induced apoptosis. Conclusion Suppression of CACNA1H activated the ERS and thus induced apoptosis in glioma cells. T-type Ca2+ channel inhibitors ABT-639 and NNC55-0396 also induced apoptosis through ERS in glioma cells. Our data highlighted the effect of CACNA1H as an oncogenic gene in human glioma.


Introduction
Gliomas are the most common central nervous system malignancies, which are characterized by low surgical resection rate, high recurrence, and poor sensitivity to chemoradiotherapy [1]. Intracellular Ca 2+ is an important signal regulating cell proliferation, differentiation, and apoptosis [2,3]. Ca 2+ channels are abnormally expressed in various tumor cells and are involved in the progression of human glioma [4,5]. T-type Ca 2+ channels are low-voltage-gated channels that are widely expressed in normal tissues, including brain, kidney, heart, and smooth muscle [6,7]. Studies have shown that T-type Ca 2+ channels are highly expressed in various types of gliomas, and their increased expression can promote tumor cell proliferation [6]. Valerie et al. found that treatment with the T-type selective antagonist mibefradil or knockdown of T-type Ca 2+ channel reduced cell viability and induced apoptosis in U251, T98G, and U87 cells [8]. At present, the specific mechanism by which T-type Ca 2+ channel regulates glioma progression remains unclear.
T-type Ca 2+ channels include three subtypes, Cav3.1, Cav3.2, and Cav3.3, encoded by CACNA1G, CACNA1H, and CACNA1I, respectively [9]. Studies have found that CACNA1H mutations exist in childhood absence epilepsy [10], generalized epilepsy with febrile seizures plus [11], and autism spectrum disorders [12], suggesting that CACNA1H plays an important role in neurological lesions. NNC-55 (T-channel inhibitor) or knockdown of CACNA1H can activate endoplasmic reticulum stress (ERS), thus increasing autophagy inhibition and apoptotic signals in CACNA1H−/− mice [13]. Tumor cells are maintained in a hypoxic, glucose-deficient, low pH microenvironment due to their rapid growth, which induces ERS and unfolded protein responses (UPRs) [14]. ERS participates in the process of tumorigenesis, development, and drug resistance by inducing autophagy, invasion, and apoptosis of tumor cells [15]. However, there is no report on whether CACNA1H participates in tumor progression through ERS. Here, we investigated this hypothesis in a glioma cell model in vitro.

Detection of cell viability
Cell-counting kit-8 (CCK8) (MedChemExpress, Monmouth Junction, NJ, USA) assay was performed to determine cell viability. The U251 cells (1,000 cells/well) were transferred into a 96-well plate. For viability detection, cells were incubated with 10 μl of CCK8 for 1.5 h at 37°C. Optical density value at 450 nm was measured using a microplate reader.

Western blot analysis
Total protein was extracted from U251 cells using RIPA buffer and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, protein samples were transferred to a polyvinylidene difluoride membrane. After blocking with 5% non-fat milk, the membrane was incubated with primary antibodies at 4℃ overnight followed by secondary antibodies at room temperature for 1 h.

Apoptosis detection
Flow cytometry was performed to detect cell apoptosis using the AnnexinV-FITC/PI kit (Beijing 4A Biotech Co., Ltd, Beijing, China) according to the operating instruction. Cell signal was collected using flow cytometry (BD, Franklin Lakes, NJ, USA). Apoptotic cells were analyzed using FlowJo software (BD Biosciences).

Data analysis
Data were analyzed using GraphPad Prism 9 and presented as mean ± SD. Student's t test and one-way analysis of variance with post-hoc Bonferroni were used for data analysis. P < 0.05 was considered statistically significant.

Knockdown of CACNA1H inhibits proliferation and induces apoptosis in glioma cells
To explore the specific role of CACNA1H in glioma cells, we inactivated CACNA1H using siRNA technology (Figure 1a) or CACNA1H inhibitor (ABT-639). ABT-639 is a Cav3.2-specific inhibitor which is less active at other Ca 2+ channels (Cav3.1 and Cav3.3). The viability decreased significantly 48 h after the knockdown of CACNA1H. The effect of ABT-639 on cell proliferation was consistent with that of CACNA1H siRNA (Figure 1b). On the contrary, the percentage of apoptosis increased significantly after CACNA1H knockdown or ABT-639 treatment (Figure 1c and d).
Then we detected apoptosis-related proteins using western blot (Figure 1e). Our results proved that the expression of

Knockdown of CACNA1H activates ERS in U251 cells
Since knockdown of CACNA1H induced tumor cell apoptosis, we further investigated whether CACNA1H was involved in apoptosis regulation through ERS pathway. As shown in Figure 2, the expression levels of ERS-related proteins were detected using western bolt. The phosphorylation level of PERK increased significantly in CACNA1H knockdown cells compared with that of control cells. The GRP78, CHOP, and ATF6 proteins were upregulated in the CACNA1H knockdown cells. The expression levels of ERS markers p-PERK, GRP78, CHOP, and ATF6 were also inhibited in ABT-639-treated U251 cells (Figure 2).

NNC55-0396 induces apoptosis through the activation of ERS in U251 cells
NNC55-0396 is a potent T-type Ca 2+ channel inhibitor, which has been found to induce apoptosis of various tumor cells in recent years. Here, we investigated its effect on U251 cells. As shown in Figure 3a, the proliferation of cells treated with NNC55-0396 decreased significantly compared with the control group. Importantly, the expression of ERS makers GRP78 and CHOP reduced significantly after the treatment with NNC55-0396 (Figure 3b-d). Bcl-2 and pro-Caspase12 were downregulated and Cleaved-Caspase12 was upregulated in NNC55-0396-treated U251 cells (Figure 3e-g). These results indicated that NNC55-0396 induces apoptosis through the activation of ERS in glioma cells.

UR906 blocked the apoptosis induced by ABT-639
Next, we investigated the effect of ERS inhibitor UR906 in U251 cells treated with ABT-639. As shown in Figure 4a, cell proliferation increased significantly after the treatment of UR906 in ABT-639-pretreated U251 cells. As predicted, the percentage of apoptosis decreased significantly after the treatment of UR906 in ABT-639-pretreated U251 cells (Figure 4b and c). UR906 also decreased the expression of Bcl-2 and pro-Caspase12, and reduced the expression of Bcl-2 in ABT-639-pretreated U251 cells (Figure 4d-g).

UR906 blocked the activation of ERS induced by ABT-639
Finally, we detected the expression of ERS markers in U251 cells. The phosphorylation level of PERK decreased significantly after the treatment of UR906 in ABT-639pretreated U251 cells. The GRP78, CHOP, and ATF6 proteins were downregulated after the treatment of UR906 in ABT-639-pretreated U251 cells ( Figure 5). These data proved that blocking ERS attenuated CACNA1H knockdown-induced apoptosis in glioma cells.

Discussion
CACNA1H encodes a low-voltage-activated calcium channel, which is expressed in thalamocortical circuits and is involved in the formation of neuronal burst firing and rhythm generation, as well as sleep and the development of epilepsy [16,17]. In recent years, the role of CACNA1H in tumors has been gradually discovered [18][19][20][21]. The genetic defect and mutation of CACNA1H involves in the progression of benign aldosterone-producing tumors [18,22]. As the cellular antenna for breast cancer-specific frequencies, CACNA1H mediates the targeted inhibition of breast cancer brain metastasis [23]. In human glioma, endostatin inhibited proliferation and migration by inhibiting the channel currents of Ca(V) 3.1 and Ca(V) 3.2 [24]. Here, we proved that the knockdown of CACNA1H inhibited proliferation and induced apoptosis in U251 cells. CACNA1H inhibitor ABT639 also suppressed proliferation and increased the percentage of apoptotic cells. Subsequently, we detected the expression of Bcl-2 and Caspase12 and found that the knockdown of CACNA1H could activate Caspase12 and inhibit Bcl-2 expression. Bcl-2 inhibits cell death caused by various cytotoxic factors and plays a critical role in tumor cell apoptosis [25]. Interestingly, Bcl-2 also inhibits the transmembrane flow of Ca 2+ . Lam et al. have reported that the apoptosis induced by the calcium pump-specific inhibitor thapsigargin can be inhibited by Bcl-2. The reason is that Bcl-2 inhibits the transmembrane flow of Ca 2+ , suggesting that Bcl-2 can regulate apoptosis by regulating the intracellular Ca 2+ [26]. Recent studies have shown that Bcl-2 family proteins interact with inositol 1,4,5-triphosphate receptor in endoplasmic reticulum (ER) to regulate its channel switch [27,28]. Anti-apoptotic proteins inhibit the excessive release of Ca 2+ from ER and support cell survival, while pro-apoptotic proteins enhance the release of Ca 2+ from ER and upregulate the concentration of Ca 2+ in mitochondria and trigger apoptosis [27,29]. However, whether Bcl-2 affects CACNA1H activity and forms feedback regulation needs further experimental study.
Studies have shown that the inhibition of CACNA1H activates ERS, thereby inducing skeletal muscle atrophy [13]. ERS can promote the processing of unfolded or misfolded proteins in the ER, which facilitates the restoration of homeostasis and maintenance of cell survival, but persistent ERS leads to UPR and cell apoptosis [30]. The main ER transmembrane proteins involved in UPR signaling are PERK, IRE-1, and ATF6 [31]. Under normal conditions, they usually bind to GRP78 in an inactive state [32]. In the presence of ERS, GRP78 dissociates from these proteins and then binds to higher affinity unfolded and misfolded proteins. At the same time, PERK, IRE-1, and ATF6 dissociated from GRP78 were activated, eventually inducing UPR and apoptosis. Our results proved that the knockdown of CACNA1H and T-type Ca 2+ channel inhibitor increased the expression of PERK, GRP78, CHOP, and ATF6, indicating the suppression of CACNA1H-activated ERS in U251 cells. Then, we found that ERS inhibitor UR906 blocked the apoptosis induced by CACNA1H inhibitor, suggesting that CACNA1H downregulation induced the apoptosis of tumor cells by activating ERS.
In addition, we also analyzed the effects of another Ttype Ca 2+ channel inhibitor, NNC55-0396, on the proliferation and ERS of glioma cells. The results confirmed that NNC55-0396 not only decreased cell activity, but also activated ERS in U251 cells. NNC55-0396 is a highly selective T-type calcium channel blocker for the Cav3.1 T-type channels, and its role in tumor cells is rarely reported. This result suggested that different subtypes of T-type Ca 2+ channels might have a universal regulatory effect on ERS and apoptosis in glioma cells, which is worthy of our in-depth study. This result also indicated that NNC55-0396 might be considered as a new small molecule with anti-cancer effect.

Conclusion
Our data highlighted the effect of CACNA1H as an oncogenic gene in human glioma. Suppression of CACNA1H activated the ERS and thus induced apoptosis in glioma cells. T-type Ca 2+ channel inhibitors ABT-639 and NNC55-0396 also induced apoptosis through ERS in glioma cells.
Author contributions: SL was responsible for doing experiments and writing the manuscript. YB assisted in the experiment. CL-L assisted in the analysis of experimental data. GM-X drafted and revised the manuscript. All the authors have read and agreed to the final manuscript.

Conflict of interest:
The authors state no conflict of interest.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.